The neutral Red probe location in large unilamellar vesicles: Influence of membrane composition and phase states

Abstract

Biological membranes are complex and dynamic self-assembled structures composed of lipids and proteins. They depict emergent properties that are heterogeneous in the membrane plane. Therefore, their study is currently one of the greatest challenges in biophysics.The versatility of spectroscopic techniques has encouraged the development of new fluorescent probes capable of sensing different membrane properties. The Neutral Red (NR) molecule is a dye that protonates at pH < 6.8 (pKa = 6.8), leading to the charged species (NRH+). NR is widely employed in molecular biology to assess cell viability and mitochondrial function, and to sense intracellular pH. However, the photophysical behavior of NR in complex media such as membranes were not systematically characterized even though of the wide application in molecular biology.In this work, the photophysical behavior of NNR (pH 8 neutral specie) and NRH+ (pH 4) was studied by employing large unilamellar vesicles (LUVs) with different compositions, membrane phase states.The results of this study show that the spectroscopic behavior of NNR is more sensitive to the membrane composition than that of NRH+. The wavelength of the maximum emission of NNR was at shorter wavelengths in liquid-disordered (Ld) membranes compared to gel or liquid-ordered (Lo) membranes. The Red-edge excitation shifts (REES) and Anisotropy magnitudes were lower in membranes in the gel or Lo, compared to membranes in Ld. The same tendency was observed for NRH+, although the spectral changes in this case were smaller, as well as the changes in the REES effect, and anisotropy magnitudes were lower related to the NR species. We suggest that NNR and NRH+ are localized in different regions of the membrane depending on the membrane phase estate. NR and NRH+ are located in more internal regions of the interface in membranes in Ld phase state, whereas in gel or Lo phase membranes, NR is located in a more external region, and NRH+ is adsorbed on the membrane surface. Consequently, the spectral signals of both, NNR and NRH+, correspond to the species distributed in different locations of the membrane in Ld compared to gel and Lo phase. Therefore, the information provided by the probe inserted in different membranes is not directly comparable.